This application relates to a method apparatus for packing tubular columns with particles, such as packing HPLC columns with media.
Liquid Chromatography is a method for the separation of compounds from a mixture by encouraging a different distribution between the solid particles in the stationary phase of a column and the liquid phase that is passed through the column. The column packing material needs to be well packed and uniformly packed to provide a consistent surface for the separations to occur. Depending on the individual compounds affinity for the column packing material compared to the affinity for the liquid passing through the column determines the difference in time that the compounds reside inside the column. After the compounds exit the column, they are either detected (analytical liquid chromatography) or the compounds are individually collected (preparative liquid chromatography). For the chromatographic separations to be carried out efficiently the column packing material bed must be uniformly and densely dispersed in the column with no channels or voids or stratification of the media that would create re-mixing of the separated compounds.
To achieve this high performance, liquid chromatography columns are generally formed by dispersing or suspending the spherical or irregular chromatographic particles in a suitable solvent creating a slurry. The slurry is placed inside the column tube and the particles are settled to form the column bed by exerting a force onto or through the slurry. This force causes the particles to sediment or “pack” into the column tube. During the packing process, the slurry solvent evacuates through a porous disk called a “frit” which is sufficiently porous to allow liquid to evacuate, but retain the chromatographic particles. As the slurry solvent is evacuated, the chromatographic particles are compressed into a uniform and dense “bed”, and the column is then ready for use.
Compressing or packing the column bed is achieved by one of two methods. One common technique of forming chromatographic beds is to produce a slurry inside the column and then using high solvent flow through the column to push the particles towards the outlet of the column. To produce these columns using the slurry technique, the chromatographic column is open at the top during the packing process and this end is attached to one end of a slurry chamber. The chromatographic media slurry fills the slurry chamber and the column tube. The outlet end of the column holds the frit that retains the chromatographic media but permits the liquid to exit the column. The inlet end of the slurry chamber is attached to a high pressure, high volume pump that forces a liquid through the chamber. The high flow liquid forces the particles to migrate towards the exit of the column and the chromatographic media is slowly compressed into a uniform and dense bed.
The slurry technique requires high-pressure pumps to provide the liquid flow until the bed is packed, which may take as long as one hour. This relatively long packing time at high flow significantly increases cost of the final column due to large volume of solvent required, and reduces manufacturing throughput. Once the chromatographic bed is formed, the packed column tube is removed from the slurry chamber and a permanent end fitting is placed onto the column. Another major disadvantage with this high pressure high flow rate slurry packing technique is that the chromatographic media is subjected to very high pressures during the packing process to form the compressed chromatographic bed but this pressure must be released when the column is removed from the slurry chamber. When the pressure is removed from the column, the compacted chromatographic media decompresses or relaxes and the chromatographic bed expands and can move within the column tube. To close the column, the excess media is removed from the top of the column and another frit and end cap is placed onto the column tube to prevent anymore shifting or expansion of the packed bed. The bed shift and subsequent removal of the excess media causes a variable and somewhat reduced bed density, particularly at the head of the column. This variation and reduction in bed density creates performance problems such as settling or voiding of the bed, particularly when the column is operated under high flow rates. This phenomenon is most pronounced on columns that have a relatively low aspect ratio (length/internal diameter) of <25. Currently users in high throughput, high volume preparative applications experience reduced lifetimes with columns prepared using this slurry technique, especially when the column lengths are short due to the lower density beds.
An alternative technique to packing a column is the use of axial compression, as generally described in U.S. Pat. No. 5,893,971. However, most chromatographic columns packed by axial compression are integrated into a single system which includes both the column and the packing apparatus. The column itself is not removable from the system itself, meaning the column is operated as part of the system. Also, the column tube is only partially filled with chromatographic media as most of the tube volume is used as a slurry chamber. Furthermore, as the packing system and column are integrated into a single unit, cost and complexity are also higher.
A newer variation of axial compression packing is described in U.S. Pat. Nos. 5,951,873 and 6,036,755. The column can be removed from the packing system by incorporating a removable spring-rod and piston that maintain active pressure on the column bed. But even this system requires a relatively long column tube, even if the desired bed length may be only ⅓ to ¼ that of the overall column length. The reason for this is that a significant portion of the overall column tube length is required simply to accommodate the uncompressed slurry prior to compression and/or the spring assembly. Thus the design suffers from long and sometime very heavy column assemblies that are inconvenient, impractical and difficult for operators to use in a typical laboratory or purification lab.
These designs are especially impractical in high-throughput purification laboratories where 4-8 columns may be run in parallel, and/or where space is limited. Furthermore, the high cost of the column hardware of this longer and more complex design limits their acceptance in high use or high-throughput environments as they become too costly to operate in large numbers.
Yet another disadvantage of some axial packed columns is that the frit diameter is much smaller than the column diameter. This reduced diameter is due to the space required for a seal or seal assembly that presses against the column wall to prevent leaking of solvent or media when high pressures are applied to the column during axial compression. This reduced diameter leaves an unused area of the column near the outer walls where the frit does not cover the chromatographic bed and the sample is not applied to this area. With short bed columns the distribution of the sample across the entire surface is critical since the bed length is very short and the sample needs to be quickly and uniformly dispensed across the surface. In slurry packed columns the frit diameter is generally equal to or greater than the column diameter.
Therefore, there is a need for more compact, disposable column designs that can be produced by axial compression packing methods and components to improve lifetime and not be cumbersome for the chemist to use.